[[Image:KAH201-KAH182-Ligation Confirmation.png|thumb|350px| Two seperate bands in each column show the vector backbone and the insert, 3200 bps and the KAH201+KAH182 (226+1675) constructed gene.Backbone, 1901 bps, Cut with EcoRI and PstI. First column: Ladder, Second collumn: 3:1 ratio, Third and fourth column: 4:1 ratio (colony A and B)]]

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[[Image:Ligation Confirmation.jpg|thumb|400px| Two seperate bands in each column show the vector backbone and the insert, 4400 bps and the KAH201+KAH182 (226+1675) constructed gene.Backbone, 1901 bps, Cut with EcoRI and PstI. First column: Ladder, Second collumn: 3:1 ratio, Third and fourth column: 4:1 ratio (colony A and B)]]

Calculations are for the ng of insert we need to get a 2:1, 3:1 and 4:1 ratios of insert molecules to 50 ng vector molecules

1 (2:1)

2 (3:1)

3 (4:1)

4 (- Ctrl)

Insert DNA

3.11

4.66

6.21

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Vector DNA

1.37

1.37

1.37

1.37

2x lgn buf (Roche)

5.48

7.03

8.58

5.0

T4 ligase (NEB)

1.0

1.0

1.0

1.0

dH2O

0.00

0.00

0.00

2.63

10.96 μL

14.06 μL

17.16μl

10μL

The incubation time for the ligation process was 15min at room temperature.

Fast transformation, 15 min on ice.

Confirm The Assembly

Two seperate bands in each column show the vector backbone and the insert, 4400 bps and the KAH201+KAH182 (226+1675) constructed gene.Backbone, 1901 bps, Cut with EcoRI and PstI. First column: Ladder, Second collumn: 3:1 ratio, Third and fourth column: 4:1 ratio (colony A and B)

Plasmid DNA

2.0 μl

EcoR1

1.0 μl

Pst1

1.0 μl

10x FastDigest buffer + green loading dye

1.5 μl

dH2O

9.5 μl

15.0 μl total

Incubate at 37°C for 10 minutes.

Make the (1%) agarose glee and add 15μl of restricted vector in one well and 10 μl of ladder.